Nutritional lactate spikes: quantitative antagonism by dichloroacetate

Nutrition Research 21 (2001) 1235–1249 www.elsevier.com/locate/nutres

Nutritional lactate spikes: quantitative antagonism by dichloroacetate Anthony W. Fox*, Catherine C. Turkel, Joan D. Buffini Cypros Pharmaceutical Corporation, 2714 Loker Avenue West, Carlsbad, CA 92008, USA Received 5 February 2001; received in revised form 24 June 2001; accepted 24 July 2001

Abstract This clinical study characterized nutritional lactate spikes, investigated their antagonism by dichloroacetate (DCA), quantitated that antagonism, and compared these with fasting lactate concentration responses. A secondary concern was the tolerability of DCA, including consequent oxaluria. We measured lactate and DCA concentrations simultaneously, using a double-blind, placebo-controlled protocol, and Schild analysis. Nutritional lactate spikes peaked at 2 mM, and lasted for about 2 h, and DCA was a non-competitive antagonist, with EC50 similar to that for reducing fasting lactate concentration. Oxaluria (up to 4-fold control) was complete by 24 –36 h, linearly related to DCA dose, and without crystalluria. Conclusions: i) Nutritional lactate spikes are non-competitively antagonized by DCA (unlike exercise-induced spikes), ii) this is a method for comparison of pyruvate dehydrogenase activating drugs, and iii) consequent oxalate loads are well-tolerated. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Lactate; Dichloroacetate; Fed/fasted; Oxalate; Antagonism

1. Introduction Nutritional spikes in plasma lactate concentration occur after both eating and intravenous glucose infusions. These spikes may be due either to increased lactate production, reduced lactate clearance [1–3] or both. Nutritional plasma lactate spikes correlate with thermogenesis and glucose utilization [4 – 6]. Lean and obese individuals differ in nutritional lactate

* Corresponding author. EBD Group, 6120 Paseo del Norte, Suites J-2–L-2, Carlsbad, CA 92009. Tel.: ⫹1-760-930-0500; fax: ⫹1-760-930-0520. E-mail address: [email protected] (A.W. Fox). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 3 3 9 - 6

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production [7]. Diabetic patients have reduced lactate clearance, regardless of ambient insulin concentration or degree of obesity [8,9]. In contrast to diabetes, hypermetabolic states (e.g. malignant hyperthermia, malaria, patients with large burns) exhibit hypoglycemia with elevated plasma lactate concentrations [10,11]. Sodium dichloroacetate (DCA; m.w. ⫽ 151 D, pK ⫽ 1.7) lowers fasting plasma lactate concentrations in human beings [12]. The molecular pharmacology of this effect resides in an inhibition of pyruvate dehydrogenase-kinase (PDH-K), which causes activation of PDH itself [13,14]. With insufficient replenishment of pyruvate production by the processes of glycolysis (e.g., after fasting to hepatic glycogen depletion), reserves of lactate are drawn upon for pyruvate synthesis; this oxidation is, of course, catalyzed by lactate dehydrogenase, and against an equilibrium which energetically strongly favors the reverse reaction [12,15, 16]. Lactate clamp studies in animals suggest that the effects of DCA are solely on the rate of lactate clearance [17], although this has not been reported in man. Lactate is a neurotoxin. There are systemic, congenital lactic acidoses [18 –22], and elevated brain concentrations of lactate after stroke [23] and head injury [24 –27]. Various multi-organ diseases are also characterized by lactic acidosis (see Discussion). Hitherto, the only specific therapies for lactic acidosis have included bicarbonate (either oral or intravenous), and the withdrawal of provocative stimuli (e.g., certain oral hypoglycemic drugs; this tactic is impossible in patients with trauma and the congenital lactic acidoses). The testing of drugs that are potentially beneficial for these various forms of lactic acidosis require normal volunteer studies that can test not only tolerability but can also handle biases in plasma lactate measurements; nutritional status is one such potential bias. DCA also perturbs lipogenesis and branched-chain amino acid metabolism [28,29], and is itself a metabolite of other drugs, e.g. chloramphenicol [30]. The effects of DCA on exercise-induced lactate spikes are minimal [31,32]. This study was conducted to discover whether nutritional lactate spikes could be blunted by DCA. A quantitative, pharmacological approach was taken to compare these effects to those on resting plasma lactate concentrations. We also took the opportunity to confirm the tolerability of intravenous DCA, and to measure the consequent oxalate excretion.

2. Methods A placebo-controlled, double-blind, ascending dose-cohort study design was used, with simultaneous estimates of venous DCA and lactate concentration. Each subject took part in only one dose cohort. The study complied with the Investigational New Drug regulations, the Good Clinical and Laboratory Practices guidelines, and the Declaration of Helsinki (as amended). Institutional Review Board approval was obtained. 2.1. Subjects Thirty-seven healthy adults (16 women and 21 men), 85–115% ideal body weight, were admitted to a purpose-built study site (GFI Pharmaceutical Services, Evansville, IN). There were three, ascending-dose cohorts with n ⫽ 12 subjects per cohort; of these 12 subjects,

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Fig. 1. Study design. Study periods are shown in chronological order from left to right, with lower bar simultaneous with upper bars. The duration of each study period is shown, but bars are not to scale. The initial period was for acclimatization and fasting. Cannula placement is shown by the arrow. The test infusion for study of nutritional lactate spikes is shown (ivi 1). After the lactate spike study, a second infusion (for oxalate tolerability) was administered (ivi 2); total urine volume was collected in 6 h aliquots.

three received placebo and nine received active treatment. Cohorts were well-matched for age, sex, and body weight. One subject was replaced for repeatedly falsifying information about participation in another study (which was a prospective exclusion criterion); as it turned out, both this subject and his replacement received placebo infusions. All subjects provided written informed consent. 2.2. Test materials DCA (Cypros Pharmaceutical Corporation, Carlsbad, CA) was dissolved in normal saline under sterile and pyrogen-free conditions. The solution was colorless, and normal saline placebo was matched for volume. Appropriate genetic [33] and general (two species, one non-rodent, 14-day intravenous; data on file) toxicology coverage had previously been demonstrated per International Conference on Harmonization guidelines, and in compliance with United States Investigational New Drugs regulations (Code of Federal Regulations 21, part 312). 2.3. Study procedures Fig. 1 illustrates the study timeline. After a fast of at least 12 h, intravenous cannulae were placed in both forearms of all subjects (one for test infusions, the other for venous sampling); heparin locks were used. Between 0.25–1 h after cannula placement, subjects received the test infusion, lasting 0.5 h. The test meal was administered between 09.40 –10.40 hrs (local time), and 1 h after the end of the infusion; this standardized breakfast contained 115 g

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carbohydrate, 30 g protein and 32 g fat, comprising 868 kcal (equivalent to 11.9 ⫾ 2.1 kcal/kg body weight for the 37 subjects). Subjects remained at rest throughout the study. A second infusion was administered between t ⫽ 8 – 8.5 h, after the lactate spike study, to increase DCA exposure and urinary oxalate excretion. 2.4. Venous blood sampling Sodium heparinized tubes were used, and placed immediately in ice. After cold centrifugation to a packed cell mass of ⬍50%, fractions of the supernatant were transferred to clean sodium heparinized tubes, and frozen at ⫺40°C for up to seven days. A staggered study procedure was used that permitted cold centrifugations to be completed within 20 minutes of sample collection. 2.5. Lactate assays Rapid thawing of aliquots, and deproteination with acetonitrile was followed by a brief cold centrifugation. Replicate supernatant samples were assayed using an automated analyzer (North Coast Clinical Laboratory Inc., Sandusky, OH). The analyzer uses the conversion of lactate to pyruvate, UV light absorption, and consumption of NAD as the reporting species. This assay had a validated range 0 –15 mM. Coefficients of variation were always ⱕ10% at ⱖ0.15 mM, and, for example, 3.3% at 1.5 mM, and 0.9% at 9.8 mM lactate, respectively. 2.6. DCA assays Aliquots of the same deproteinated supernatants were extracted into acidic organic solvent, and derivatized with diazomethane. Gas-liquid chromatography was used with an electron-capture detector (Wisconsin Analytical Research Services, Madison, WI). Peaks were symmetrical, with area under the curve linearly related to concentration for [DCA] 0.2– 400 ␮g/mL. The limit of quantitation was ⱖ0.05 ␮g/mL. 2.7. Oxalate assays Urine fractions (each 6 h interval) were homogenized and sampled. Assays used a proprietary automated procedure (North Coast Clinical Laboratory, Sandusky, OH). Absolute amount of oxalate excreted in each fraction (mg) was calculated from concentration and volume. 2.8. Statistical methods Area under the curve for lactate concentration vs. time was estimated using a trapezoidal rule (the end of the curve was prospectively defined as the return of mean lactate concentration to pre-prandial values, i.e., 2.5 h, see below). Effects of DCA on resting plasma lactate concentration were quantified as the difference between mean baseline concentration (i.e., fasting, pre-infusion), and the lowest observed plasma lactate concentration during the 2 h

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interval before the test meal. Maximal blunting of the nutritional lactate spike was prospectively defined as a lactate concentration equal to that immediately before the test meal. Unless stated, mean ⫾ sem values are shown for n ⫽ 10 placebo- and n ⫽ 9 active-treated subjects. Student’s t tests were two-tailed, using the unpaired equation, which does not assume equal variance between groups. Schild analysis [34] requires that agonist dose- or concentration response curves are parallel in the absence and presence of a truly competitive antagonist. Usually, only unequal response sizes can be measured with and without the antagonist being present. The dose ratio is then found for constant response size (usually 50% maximal), by linear interpolation of all responses between 20% and 80% of maximal (i.e., linear interpolation of EC50, using all observed values between EC20 and EC80). The slopes of concentration-response curves can be compared for parallelism by measuring Log (EC80/EC20) ⫽ Log EC80 ⫺ Log EC20, with an assumption that this segment of the curve is straight. The potency of a known concentration of antagonist is measured by the degree of shift of an agonist concentration-response curve from its control. Potency of antagonists are compared by Schild plots, i.e.: Log10共Dose ratio ⫺ 1兲 ⫽ ⫺a.Log10关antagonist兴 ⫹ c, where a is the slope of the plot and c is a location parameter. For competitive antagonists, this plot is linear, and has unit slope. The x axis intercept is the Schild constant (or pA2). The pA2, thus represents the negative logarithm of the concentration of antagonist that causes responses between EC20 and EC80 to require twice as much agonist. For many competitive antagonists, the Schild constant correlates closely with the equilibrium Ki at the receptor [35]. In this study, each subject took part in only one dose cohort. When the observed responses are known to lie between the EC20 and EC80, then a putative EC50 can be found from each response measurement by linear extrapolation. This requires an assumption that agonist concentration-response curves are parallel (i.e., Log (EC80-EC20) is assumed to be constant), but the precise value of the slope is not needed: An arbitrary value may be chosen, as long as it is used uniformly. A dose ratio can be calculated from the putative EC50 values, and dose ratios and x axis intercepts on the Schild plot may then be found. This corollary of Schild analysis cannot rigorously fulfill all the criteria for identification of a competitive antagonist. Similar x intercepts for different antagonist concentrations are consistent with but not conclusive for competitive antagonism. However, if antagonists fail to fulfill one or more of the criteria for true competitiveness, then divergent x axis intercepts result, and non-competitive antagonism can be reliably inferred.

3. Results 3.1. Baseline fasting plasma lactate concentrations Prior to the test infusions, fasting plasma lactate concentration for the study population was (n ⫽ 37) 0.95 ⫾ 0.09 mM. The mean value for women (0.66 ⫾ 0.09 mM, n ⫽ 16) was

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Fig. 2. Fasting, resting plasma lactate concentrations before (open bars) and 2 h after (closed bars) placebo or sodium dichloroacetate infusion. Mean ⫾ sem values are shown, n ⫽ 9 or 10 subjects per treatment group. *p ⬍ 0.05 compared with pre-infusion value.

lower than for men (1.17 ⫾ 0.13 mM, n ⫽ 21; p ⬍ 0.05). There were no significant correlations between body weight, and fasting plasma lactate concentration (R ⫽ 0.026, ⫺0.011, and 0.31 for men, women and the whole study population, respectively). 3.2. Effect of test infusions on fasting plasma lactate concentration Just prior to the test meal, and 1 h after placebo infusion, fasting plasma lactate concentrations were 0.98 ⫾ 0.12 mM. For the 30, 60 and 100 mg/kg treated subjects, respectively, plasma DCA concentrations just prior to the test meal were 66.7 ⫾ 10.1, 175 ⫾ 8.8, and 267 ⫾ 13.3 ␮g/mL; these were inversely associated with fasting plasma lactate concentrations of 0.48 ⫾ 0.08, 0.42 ⫾ 0.07, and 0.32 ⫾ 0.07 mM (p ⬍ 0.05 for any active treatment group vs. placebo; no statistical differences were found among active treatment groups; Fig. 2). 3.3. Quantitation of nutritional lactate spike Fig. 3 shows nutritional lactate spikes for placebo-treated subjects, with peak lactate concentrations (Lactatemax) of 2.03 ⫾ 0.25 mM and Tmax at 0.5h (p ⬍ 0.01 compared with immediately pre-prandial). Plasma lactate concentrations returned to pre-prandial values at 2.5 h after the test meal. Unadjusted areas under the curve (AUC) between t ⫽ 0 and 2.5 h after the test meal were 4.23 ⫾ 0.33 mM.h. After subtraction of rectangles representing fasting plasma lactate concentration and spike duration (2.5 h) from unadjusted AUC,

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Fig. 3. Quantitation of nutritional lactate spike. Subjects ate the standardized test meal between t ⫽ 0 and 0.33 h, having received a placebo (normal saline) infusion 1.5 h before. Polygons A (left hand panel, unadjusted lactate concentrations) and A1 (right hand panel, baseline lactate concentration subtracted) did not differ in area (mean ⫾ sem values shown, n ⫽ 9 subjects).

incremental lactate spikes were 1.81 ⫾ 0.20 mM.h. These AUCs were consistent with those calculated from earlier reports (see Table). 3.4. Effect of DCA on nutritional lactate spikes The Cmax for DCA was 50.3 ⫾ 8.9, 149 ⫾ 7.7, and 257 ⫾ 16.1 ␮g/mL, for the 30, 60 and 100 mg/kg dose groups, respectively. Lactatemax was 1.06 ⫾ 0.22, 0.90 ⫾ 0.14, and 0.44 ⫾ 0.07 mM for the 30, 60, and 100 mg/kg doses, respectively (Fig. 4; p ⬍ 0.01 for any active group vs. placebo, p ⬍ 0.03 for 30 vs. 100 mg/kg, otherwise, no differences between active treatment groups). DCA did not alter nutritional lactate spike duration. Adjusted lactate spike AUC was also inversely dose-ordered: 0.78 ⫾ 0.30, 0.56 ⫾ 0.21, and 0.02 ⫾ 0.12 mM.h for the 30, 60 and 100 mg/kg doses, respectively (p ⬍ 0.04 between any pair of active treatment groups). The concentration-response relationship for lactate spike blunting was similar to that for fasting lactate concentration (apparent mean EC50 values of 0.55 and 0.66 mM DCA for fasting lactate concentration and nutritional lactatemax, respectively; Fig. 5). 3.5. Schild analysis for competitive antagonism Fig. 6 shows the putative Schild plots using lactatemax as the response. The plot using the mean values from all three DCA treatment groups had slope ⬍⬍1.0. Furthermore, the

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Fig. 4. Circulating concentrations of dichloroacetate (upper panel) and lactate (lower panel, following administration of test meal between t ⫽ 0 and 0.33 h. Subjects had received intravenous infusions of dichloroacetate 1.5 h before at doses of 30 (triangles), 60 (closed boxes) or 100 (open boxes) mg/kg. Mean ⫾ sem values shown, n ⫽ 9 subjects per treatment group.

putative pA2 values, assuming unit slope, diverged widely from that for all three dose groups. These observations, for the mean values, would be the same if all 27 subjects values were plotted separately. 3.6. Tolerability All subjects completed the experiment, without serious adverse events. Three subjects (all after receiving the highest initial dose, 100 mg/kg/0.5 h, and for about 15– 45 minutes after the test infusions began) reported somnolence as an adverse event. However, no subject slept, and the study staff did not observe any reduction in rousability. Other adverse event types obeyed no dose-related trend, being reported after both placebo- and active-treatments. In some cases (e.g., headache), adverse events were attributed by subjects to withdrawal from food or caffeine.

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Fig. 5. Concentration-response relationships for two effects of intravenous dichloroacetate (DCA). Lactate and dichloroacetate concentrations were measured simultaneously. Mean ⫾ sem values are shown for response (vertical bars) and plasma DCA concentration (horizontal bars), with n ⫽ 9 or 10 subjects in each treatment group. Open boxes: Resting, fasting plasma lactate concentration. Response was defined as % maximal, calculated as: 100 ⫺ 共lowest observed plasma [lactate]/control mean plasma [lactate]兲 ⫻ 100 Closed boxes: Blunting of nutritional lactate spikes. Spike height (mM) for each subject was defined as the difference between peak and immediately pre-test meal lactate concentrations. Response (%) was defined as: 100 ⫺ 共observed lactate spike/placebo-associated lactate spike兲 ⫻ 100

Urinary oxalate excretion (0 –36 h) was 42.6 ⫾ 2.0 mg for placebo-treated subjects. After DCA administration, this increased in linear fashion to 88.8 ⫾ 5.1, 139 ⫾ 11, and 197 ⫾ 13 mg, or a mean of 1.01–1.07 mg for each mg/kg DCA administered. The incremental oxalate excretion was 90.1–95.2% complete in the first 24 h of the 36 h collection (Fig. 7). There was no crystalluria on microscopy of urine samples.

4. Discussion Nutritional lactate spikes lasted 2.5 h, peaked at about 2 mM in control subjects, and were depressed in a concentration-dependent fashion by circulating DCA, whether analyzed by peak concentration, unadjusted AUC, or adjusted AUC. The EC50 values for DCA were 0.55 and 0.65 mM for reduction of fasting lactate and nutritional lactate concentrations, respec-

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Fig. 6. Schild analysis of the antagonism of the maximal nutritional lactate spike height (mM) by dichloroacetate: No evidence for a competitive model. The solid line represents the linear regression for the mean points of the three treatment groups. This line has slope ⬍⬍1, and low x intercept (arrow). Dashed lines: theoretical pA2 values with an assumption of unit slope. Note that the former and latter pA2 estimations diverge widely. These observations are incompatible with a competitive model of antagonism by DCA of lactate spikes.

tively. Schild analysis was obviously incompatible with a competitive model of DCA antagonism of the nutritional lactate spikes. Consequent oxalate excretion accounted for about 1.2% of the mass of DCA administered, was linearly related to DCA dose, mostly complete within 24 h of the first infusion, wholly complete within 36 h, and without crystalluria. Tangentially, our measurements of fasting lactate concentrations and nutritional lactate spikes (in placebo-treated subjects) were comparable to those in previous reports, thus validating our handling of lactate samples, which can be labile. Nutritional and exercise-induced lactate spikes differ in several ways. First, nutritional spikes can evidently be antagonized with DCA, while exercise-induced lactate spikes, with greater lactatemax can be blunted to only a minor degree, e.g., not at all by 35 mg/kg i.v. DCA, or by about only 10% of lactatemax after a 50 mg/kg DCA infusion [32,38]. This differential sensitivity to DCA might reflect differential access of DCA to the tissues generating such spikes in the plasma, i.e., the liver and skeletal muscle. Maximal physiological activation of pyruvate dehydrogenase by maximal anaerobic stress, is more likely at exhaustion, leaving little scope for an effect of DCA. “Splanchnic” and “peripheral” lactate pools have been previously described, but seem to be an over-simplification of the real situation given the differential effects of DCA [2,3,36]. The ability to measure, indepen-

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Fig. 7. Mean ⫾ sem urinary excretion of oxalate (mg) after intravenous administration of two infusions of dichloroacetate at t ⫽ 0 – 0.5 and 8 – 8.5 h. Upper panel: oxalate excretion between t ⫽ 0 and 24 h. Lower panel: oxalate excretion between t ⫽ 24 and 36 h.

dently, lactate production and lactate clearance, perhaps using a lactate clamp technique could resolve this issue. However, this has, so far, been reported only in rats [16], and clear differences in lactate metabolism exist among both familiar [36], and exotic [37] species. It is therefore difficult to interpret mechanistically these differences between types of lactate spike. It is hazardous to extrapolate from physiological to pathological states of elevated plasma lactate concentration. Patients with pathological states causing lactic acidosis usually exhibit plasma lactate concentrations that are higher than those achieved in normal subjects after a meal. Exercise exhaustion is associated with lactate concentrations ⱖ7 mM [32,38]. Malignant hyperthermia and patients with sepsis or burns are classic examples of muscle hypermetabolism causing elevated circulating lactate concentrations often up to 12 mM; the former is rarer and harder to study [10,38]. In one study of ICU patients, DCA was shown to cause reduction in circulating lactate concentration, although this was not associated with any improvement in morbidity or mortality [28]. Furthermore, a recent study has demonstrated that neither the pharmacokinetics nor pharmacodynamics of DCA in patients with

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head trauma can be predicted from studies in uninjured subjects [39]. The potential for beneficial effects in patients infected with malaria, which kills two million children annually worldwide, and where lactic acidosis is a strong predictor of mortality, is also under investigation [11,40,41]. Patients with chronic fatigue syndrome and mitochondrial myopathy typically exhibit resting, fasting plasma lactate concentrations of 1.5–2 mM, i.e., concentrations similar to lactatemax observed after the test meal in this study [42]. It might be a worthwhile experiment to expose such patients to modest DCA doses, in order to test whether circulating lactate concentration correlates with their subjective symptoms. Long-term therapy would, of course, require extensive preliminary toxicological coverage, and close monitoring for the potential of DCA to cause peripheral neuropathy [43]. Some cases of congenital lactic acidosis (CLA) also respond to DCA, administered orally or intravenously [18,20]; at 25 mg/kg DCA b.i.d., p.o., these children exhibit no oxalate crystalluria, and can avoid peripheral neuropathy with dose limitation and thiamine supplementation (R. Haas MD, University of California at San Diego, personal communication). Various halogenated alkanes and acetophenones activate pyruvate dehydrogenase [13,14, 44]. These classes of drugs mostly await adequate toxicology studies before they can be tested in man. However, human tests will inevitably begin with normal volunteer studies. Such studies can be leveraged into providing more information than orthodox explorations of tolerability and pharmacokinetics. The pharmacodynamics of these drugs can also be explored, and compared, using even first-in-man protocols, such as this. This is also consistent with the ethical imperative to obtain as much information as possible from every human exposure to an investigational drug. This imperative is especially important in the case of normal volunteers, for whom there is no possibility of clinical benefit. Oxalate is freely filtered by the glomerulus, being wholly ionized at physiological pH (pKa,b ⫽ 1.23 and 4.19). Oxalate competes with other acids for active reuptake pumps, especially that characterized for para-amino hippurate, and the molecule is not useful for measuring glomerular filtration rate. The largest DCA administration in this study caused about a four-fold increase in urinary oxalate excretion. This compares with Asiatic diets typically containing four-fold greater oxalate content than western diets, although the absolute bioavailability of oral oxalate is ⬍15%, probably because of calcium chelation in the gut [45]. In future studies, using multiple DCA infusions, the potential for oxalate cumulation must be examined, although its free filtration, and the presumably saturable nature of its active reuptake, are both favorable features for tolerability. Sulfite coadministration might also be a feasible way to limit the conversion of DCA into oxalate [46]. In summary, we have quantitated nutritional lactate spikes and demonstrated that they are antagonized by DCA. This antagonism is graded, quantifiable and non-competitive. The EC50 for this antagonism appears to be similar to that for the effect of DCA on fasting lactate concentration. The penalty to be paid in terms of increased urinary oxalate excretion appears to fall within the redundancy of function in the normal kidney within this dose range, and also within the range of human dietary oxalate challenge. The ability to perturb lactate concentrations in opposite directions may be a useful tool, in the absence of lactate clamp or other more complex methods, for comparisons of PDH activating drugs.

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Acknowledgments We thank the volunteers. Dr. Fred Hoehler, Bob Nicora and Maura Neichin RN of SDCRA (now Quintiles Pacific), Carlsbad, CA helped with data management, quality assurance and statistical advice. Dr. Randal O’Rourke of WARS (now Covance), Madison, WI was in charge of the study site and clinical safety. Dr. Randal Stoltz (GFI, Evansville, IN) helped with some of the assays. We also thank Dr. S. Krishna (St. George’s Hospital, London, UK) for some helpful comments.

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